Publications

4482

Abstract Background ‘ Candidatus Liberibacter solanacearum’ (Lso) is a phloem-limited bacterial pathogen causing significant diseases in solanaceous crops. In the United States, haplotypes A and B are transmitted by the potato psyllid Bactericera cockerelli . We previously identified differences in their acquisition and transmission between adults and nymphs. The present study characterized the dynamics of LsoA and LsoB acquisition and transmission by nymphs and examined the transcriptional responses of the nymphal gut upon their acquisition. Methods and results Nymphs were exposed to LsoA- or LsoB-infected plants for 1, 3, 5, or 7 days to measure the bacterial accumulation and for 8 days to assess the transmission efficiency following sequential inoculation of tomato plants. Quantitative PCR showed that LsoB accumulated to higher levels than LsoA after 3 days of acquisition. Following the sequential inoculation, LsoB was transmitted earlier than LsoA indicating a shorter latency period. RNA-seq analysis of the guts following a 1- and 5-day acquisition access periods revealed a greater transcriptional regulation at 5 days than at 1 day. Furthermore, the responses were haplotype-specific: LsoA primarily affected genes involved in protein translation, ER stress, and cell cycle regulation, whereas LsoB regulated genes involved in autophagy, apoptosis, and immune pathways. Conclusions This study revealed haplotype-specific gene regulation potentially leading to LsoB being transmitted more efficiently by psyllid nymphs.

ABSTRACT Citrus Huanglongbing (HLB), caused by ‘ Candidatus Liberibacter asiaticus’ ( Ca Las), is the most devastating disease affecting the global citrus industry. Here, we reported that the Ca Las effector SDE70 promotes HLB pathogenicity by targeting the citrus ubiquitination pathway. Transgenic expression of SDE70 in Wanjincheng orange ( Citrus sinensis Osbeck) accelerated early Ca Las proliferation, aggravated HLB symptoms, and increased susceptibility to citrus canker induced by Xanthomonas citri subsp. citri ( Xcc ). These results demonstrate that SDE70 functions as a broad‐spectrum suppressor of citrus immunity. Mechanistically, SDE70 physically interacts with CsRUB2, a citrus ubiquitin‐related protein. Furthermore, CsRUB2 overexpression in Wanjincheng oranges reduced resistance to HLB but enhanced resistance to citrus canker. Both SDE70 and CsRUB2 elevated salicylic acid (SA) and hydrogen peroxide (H 2 O 2 ) levels in transgenic plants while lowering methyl salicylate (MeSA) levels. CsRUB2 also decreased jasmonic acid (JA). In contrast to the suppressive effect of SDE70, CsRUB2 enhanced the transcription of citrus immunity genes. Transient expression assays further demonstrated that the SDE70–CsRUB2 interaction dysregulates citrus immunity by perturbing SA, MeSA, JA, and H 2 O 2 signals. These findings provide a theoretical basis for understanding citrus– Ca Las interactions and breeding citrus varieties with broad‐spectrum resistance to both HLB and citrus canker.

Abstract ‘Candidatus Mycoplasma haemolamae’ (CMhl) is a hemotropic bacterium infecting South American Camelids (SAC), whose epidemiology and clinical significance are still not fully elucidated. This study investigated CMhl by qPCR in blood samples of alpacas ( Vicugna pacos ) and llamas ( Lama glama ) in Italy in 2021–2024, characterizing CMhl haplotypes based on partial 16 S rRNA sequences and defining haematological findings in a subset of CMhl-infected animals. Statistical analysis was performed to detect potential risk factors associated with CMhl positivity. Out of 206 animals (200 alpacas and 6 llamas), CMhl was detected by qPCR in 88 (42.7%) animals. CMhl haplotype #1 and #2 were detected in 42.2% and 0.5% of the animals, respectively. Binary logistic regression analysis showed that higher CMhl positivity was significantly associated with larger herd size ( P  = 0.011), Northern Italy area of origin ( P  = 0.031) and warmer seasons ( P  = 0.003). The detection of two CMhl haplotypes confirms the high diffusion of CMhl in SAC farmed in Italy. Results confirm that CMhl usually causes subclinical infections, suggesting that antimicrobial treatment is likely not necessary based on qPCR positivity alone. Results focusing on the association of CMhl positivity with warm temperatures and large herd size suggest the need of further studies on the genetic features, epidemiology and immune response in CMhl-positive SAC from Italy, with emphasis on understanding potential vector transmission.

The Asian citrus psyllid, Diaphorina citri, transmits the putative causal agent of citrus greening disease, ‘Candidatus Liberibacter asiaticus’. The transmission occurs in a propagative circulative manner. Specific protein-protein interactions are required for the accomplishment of the transmission process. We employed the far-Western technique, also known as protein overlay assay, and mass spectrometry, to identify D. citri proteins that specifically recognize membrane proteins of ‘Ca. L. asiaticus’. Whole-body protein extracts of D. citri were separated by electrophoresis and blotted onto polyvinylidene difluoride (PVDF) membranes. The membranes were overlayed with sonicated leaf midrib extracts from ‘Ca. L. asiaticus’-infected citrus plants. The protein complexes were detected using different antibodies against ‘Ca. L. asiaticus’ membrane proteins and peptidoglycans associated with lipoproteins. By comparison, spots were extracted from a polyacrylamide gel for LC-MS-MS analysis. Identified proteins included ATP synthase alpha and beta subunits, actin, alpha- and beta-tubulins, transitional endoplasmic reticulum ATPase TER94, 78 kDa glucose-regulated protein, and arginine kinase. These findings emphasized the involvement of energy machinery and muscle proteins in the ‘Ca. L. asiaticus’-D. citri interactions. Understanding the pathogen-vector interactions will lead to better-designed control strategies based on the interference with specific interactions and/or blocking transmission.

ABSTRACT Insect‐vectored pathogens pose a significant threat to global agriculture. The colonization efficiency of pathogens in vectors plays a central role in these pathosystems, yet studies of the factors that affect this aspect are limited. This study investigates the genetic and microbial symbiont factors influencing the susceptibility of Diaphorina citri to Candidatus Liberibacter asiaticus ( C Las), the pathogen causing citrus greening disease (huanglongbing). Through a microbiome Genome Wide Association Study (mGWAS) based on 16S amplicon sequencing and genomic resequencing of 120 D. citri individuals from six populations, we identified 79 SNPs significantly associated with the relative abundance of C Las within insects. Additionally, some of these SNPs were also associated with the relative abundance of Candidatus Profftella armature, a key endosymbiont of D. citri . SNPs in the regulatory region of gene Dcitr04g11610.1 led to its overexpression in C Las‐susceptible D. citri , and C Las infection further elevated its expression. Conversely, RNAi knockdown of Dcitr04g11610.1 reduced C Las infection rates and abundance, accompanied by increased abundance of Profftella . Phylogenetic analysis revealed Dcitr04g11610.1 ’s high homology to Major Facilitator Superfamily‐type transporter SLC18B1 proteins, suggesting a role in C Las polyamine utilization. These findings highlight the importance and potential interplay of insect genetics and symbiotic microbiota in insect‐vectored plant pathogen systems.

Abstract The African citrus triozid (ACT), Trioza erytreae (Del Guercio, 1918) (Hemiptera: Triozidae), is a significant pest of citrus in southern Africa serving as a vector of African citrus greening disease associated with the phloem-limited gram-negative bacterium “Candidatus Liberibacter africanus” (CLaf). ACT can also vector another Liberibacter species, “Ca. Liberibacter asiaticus” (CLas) associated with Asian citrus greening. Given the confirmed presence of CLas on the African continent, efforts to monitor populations of ACT as well as associated liberibacters in citrus environments have been enhanced since 2021. Trapping and active collections in citrus surroundings indicated the presence of two triozid species with similar morphology to ACT and five other triozids which were morphologically distinct. This study aimed to characterise the diversity of triozid species around citrus and determine their association with liberibacter. Morphological characters and nucleotide sequence phylogeny of the mitochondrial cytochrome c oxidase I (COI) gene region supported the distinction of seven triozid species including ACT. CLaf was only detected in ACT. None of the other triozid species had any liberibacter species associated with them. Our results suggest the presence of other triozid species in other African countries, which probably were misidentified as ACT. Our findings highlight the need for a comprehensive taxonomic revision of triozids associated with citrus. Integrating morphological and molecular characters in a revised classification will be essential for accurate identification of ACT. Molecular confirmation is needed for confidence in distinguishing ACT from closely related non-pest species.

ABSTRACT Cable bacteria conduct long-distance electron transport in sediments but are not yet isolated in pure culture. We report the metagenome-assembled genome of Candidatus Electrothrix sp. NPCB-01 from Newport Bay, California. This 3.46-Mb genome encodes sulfur oxidation, nitrogen and carbon metabolism, and nickel homeostasis genes, expanding resources for these electroactive microbes.

Welsh onion (Allium fistulosum) is an important aromatic vegetable crop, particularly in Asia (Kim et al. 2023). In November 2025, two-month-old Welsh onion plants showing leaf proliferation, little leaf, and yellowing symptoms were found at low frequency (< 1%) in Fangyuan Township, Changhua County, Taiwan. The plot was around 880 m 2 in size and about 7,200 clumps were grown. Five symptomatic and three symptomless plants were sampled (bagged separately). The plants’ basal leaf tissues were subjected to DNA extraction and PCR analysis. DNA was extracted using a Synergy 2.0 Plant DNA Extraction Kit (OPS Diagnostics) using filter tips, and the DNA samples’ quality was verified by successful amplification of a plant 26S rDNA fragment (0.7 kb) using primer pair 28KJ/28C (Cullings 1992). Nested polymerase chain reaction (PCR) assays using phytoplasma 16S rDNA-specific primers were conducted. The nested primer pairs used were P1/P7 and fU5/rU3 (Lorenz et al. 1995), respectively. DNA of periwinkle infected with periwinkle leaf yellowing phytoplasma served as a positive control and no-template controls (nuclease-free water) were included in both PCR rounds. In both tests, only DNA from the five symptomatic plants produced the expected amplicons (1.8 kb and 881 bp for the outer and inner assays). The products from the first and second rounds of PCR were sequenced and assembled. Sequencing chromatograms of the PCR products had single, well-resolved peaks across all positions in all the samples, indicating that only one strain was present. Near-full-length (1,466 bp) 16S rDNA sequences of all five samples were identical to that of a reference ‘Candidatus Phytoplasma australasiaticum’-related strain, NCHU2014 (accession no. CP040925, bp 537,765 to 539,230; 16SrII-A; Rodrigues Jardim et al. 2023), and a representative sequence has been deposited in GenBank (accession no. PX696818). A 1,248 bp fragment (accession no. PX696818, bp 84 to 1,331) of the detected 16S rDNA sequence was excised for analysis using the web tool iPhyClassifier (https://plantpathology.ba.ars.usda.gov/cgi-bin/resource/iphyclassifier.cgi), and the virtual RFLP patterns obtained were identical to the reference pattern of 16SrII-A (accession no. L33765; similarity coefficient 1.00). Additional PCR tests targeting the protein translocase gene secY and the elongation factor Tu gene (tuf) of 16SrII phytoplasmas were respectively conducted with the infected samples using primer pairs SecYF1(II)/SecYR1(II) and TUF-II-F1/TUF-II-R1 (Al-Subhi et al. 2017; Lee et al. 2010). The secY (accession no. PX737873; 1,263 bp) and tuf (accession no. PX737874; 1,183 bp) sequences were again found identical to those of the reference strain NCHU2014 (16SrII-A phytoplasma; accession no. CP040925, bp 192,846 to 194,108 and bp 139,150 to 140,332). In Taiwan, 16SrII phytoplasma has been reported affecting various plants, such as seseme and purple coneflower (Rodrigues Jardim et al. 2023; Tseng et al. 2014). In A. fistulosum, however, phytoplasma infection has only been associated with group 16SrI (Cho et al. 2019). The present study is the first report of a 16SrII-A phytoplasma affecting Welsh onion and the findings expand the understanding of the width of the subgroup’s host range in the island. Since Welsh onion is an important culinary crop, and the disease significantly affects its morphology and commercial value, ongoing surveillance of its spread in other regions is warranted.